A molecular understanding of the genetic basis for brain activity will inevitably improve medical interventions relating to myriad neurological conditions and disorders. A critical step in peeling back the molecular inner-workings of the brain will be to assay the brain transcriptome, the messenger RNA complement within individual cells that controls function at multiple levels of organization-cell, synapse, tissue, nd brain region. Current methods for harvesting mRNA from cells in morphologically complex neuronal tissue are invasive and prone to contamination. Biological goals of this proposal are to catalog differences in neuronal transcriptome variability in different hippocampal regions in the live mouse brain slice model, and then to assess temporal and spatial aspects of transcriptome changes in response to genetic perturbation through the use of conditional CREB KO and CREB Ser133Ala knockin mice. CREB is a transcription factor that modulates various cAMP-coordinated behaviors including learning and memory, drug addiction and fear conditioning. Our goal in these model systems will be to assess, for the first time, the gene expression targets of CREB in vivo where the microenvironment will likely be an important contributor to the selection of CREB targets. To address these challenges, we have developed a light-activated transcriptome in vivo analysis (TIVA)-tag that shows extreme promise for harvesting mRNA from individual cells, in cultured neurons and rat hippocampal brain slices. TIVA-tags are caged oligonucleotides, with a poly-U capture strand that binds the poly-A tail of cellular mRNA upon photoactivation. These studies motivate the development of TIVA-tags with even greater in vivo functionality. We propose the optimization of many features in the TIVA-tag design (Aim 1), as well as development of caged ruthenium photosensitizer (Ru)-TIVA-tags suitable for 2-photon activation (Aim 2). Finally, we will develop multiplexing strategies for deep-tissue mRNA harvesting by tuning the ligands on Ru-TIVA and by developing poly-TIVA, a complementary caging technology for delivering poly-U capture strands (and other oligonucleotides) using nanoscale, cell-permeable, near-IR-responsive polymersome vesicles (Aim 3). Ru-TIVA variants and poly- TIVA will extend the capability to capture simultaneously mRNA from multiple individual cells within a tissue, or from single cells at multiple time points (i.e., longitudinal studies). These tandem technologies should make it possible to assess and potentially reprogram cellular function in the brain.